Project Summary The definitive endoderm (DE) emerges during gastrulation as an epithelial sheet. This single cell sheet, which produces the entire gut tube and associated organs, is patterned and differentiates into organ domains in response to inductive cues provided by adjacent mesoderm-derived tissues. During induction, the hepatic progenitor, the hepatoblast, emerges from the DE as a group of thickened cells that express hepatic markers. The nascent hepatoblasts form a liver bud that will invade adjacent mesodermal tissue. Once invasion is complete, a single midline rostral lobe and two smaller bilaterally symmetric caudal lobes are evident. Although hepatoblasts and their associated mesoderm have each been regarded as uniform populations, we have discovered remarkable heterogeneity in both. Firstly, hepatoblast arise from two spatially distinct DE populations that each contributes uniquely to the liver bud. The ventral midline progenitors contribute mainly to the hepatoblasts that populate the anterior liver bud while the bilaterally symmetric lateral progenitors produce hepatoblasts that contribute to the posterior liver bud. Surprisingly, each progenitor has unique requirements for this process and is each associated with different supportive mesenchyme upon induction. For example, FGF signals are required to induce the sinus venosus bounded anterior hepatoblasts, while the septum transversum mesenchyme bounded posterior hepatoblasts require BMP signaling to be induced Furthermore, recent fate mapping from our group demonstrates that the anterior hepatoblasts mainly contribute to the caudal lobes while posterior hepatoblasts mainly contribute to the rostral lobe. Finally unpublished histological and molecular data suggests that the mesenchyme supporting the rostral and caudal lobes are distinct tissues. Together these observations suggest that normal development follows two routes for generating hepatoblasts. We hypothesize that the heterogeneity generated during early hepatic development is essential for normal liver function and that it aids in generating the heterogeneity recently identified in adult hepatocyte and cholangiocyte populations. Guided by the new developmental framework outlined above, the aims of our proposal use our expertise in embryology and the power of mouse genetics to further delineate this heterogeneity and to identify how it contributes to normal development. In Aim1 we propose to use conditional knock-out strategies to assess how FGF and BMP signals contribute to hepatoblast induction in vivo. In Aim2, we use an ex vivo approach and novel mouse reporters in vivo to uncover the developmental origin and adult contribution of the rostral and caudal lobe mesenchyme as well as determine the molecular signature of the rostral and caudal lobe hepatoblasts. The long-term goals are to understand how developmental heterogeneity contributes to key adult responses such as homeostasis and regeneration and to use these developmental insights to produce functional hepatic tissues in vitro.